Antifungal Mechanism of Action of Lactoferrin: Identification of H ⁺ -ATPase (P _3A -Type) as a New Apoptotic-Cell Membrane Receptor

Abstract

bHuman lactoferrin (hLf) is a protein of the innate immune system which induces an apoptotic-like process in yeast. Determination of the susceptibility to lactoferrin of several yeast species under different metabolic conditions, respiratory activity, cytoplasmic ATP levels, and external medium acidification mediated by glucose assays suggested plasma membrane Pma1p (P 3A -type ATPase) as the hLf molecular target. The inhibition of plasma membrane ATPase activity by hLf and the identification of Pma1p as the hLf-binding membrane protein confirmed the previous physiological evidence. Consistent with this, cytoplasmic ATP levels progressively increased in hLf-treated Candida albicans cells. However, oligomycin, a specific inhibitor of the mitochondrial F-type ATPase proton pump (mtATPase), abrogated the antifungal activity of hLf, indicating a crucial role for mtATPase in the apoptotic process. We suggest that lactoferrin targeted plasma membrane Pma1p H ؉ -ATPase, perturbing the cytoplasmic ion homeostasis (i.e., cytoplasmic H ؉ accumulation and subsequent K ؉ efflux) and inducing a lethal mitochondrial dysfunction. This initial event involved a normal mitochondrial ATP synthase activity responsible for both the ATP increment and subsequent hypothetical mitochondrial proton flooding process. We conclude that human lactoferrin inhibited Pma1p H ؉ -ATPase, inducing an apoptotic-like process in metabolically active yeast. Involvement of mitochondrial H ؉ -ATPase (nonreverted) was essential for the progress of this programmed cell death in which the ionic homeostasis perturbation seems to precede classical nonionic apoptotic events. L actoferrin (Lf) is a 77-kDa iron-binding glycoprotein of the transferrin family of proteins and an important effector molecule of innate immunity with antimicrobial activity in mammalian mucosal fluids (reviewed in reference 1). We have previously reported that human lactoferrin (hLf) induces apoptosis-like cell death of the pathogenic opportunistic yeast pathogen Candida albicans, demonstrating also that K ϩ channel-mediated K ϩ efflux is a common apoptotic event in yeast and metazoan cells (2). At present, the mechanism of action by which lactoferrin and other antimicrobial peptides induce apoptosis-like processes in yeast remains unknown (reviewed in reference 3). However, we have previously identified in bacteria the proton-translocating ATPase (H ϩ -ATPase) as the molecular target of lactoferrin, suggesting that the subsequent perturbation of the proton gradient (⌬pH) and intracellular pH (pH i ) will lead to cell death in vitro (4). This finding revealed lactoferrin as a natural extracellular inhibitor of the proton translocation mediated by H ϩ -ATPases, suggesting that the possible inhibition of a similar target also explains its antifungal activity. In this hypothetical case, the antifungal effect of lactoferrin might be due to altered pH homeostasis, as reported for bacteria (4).In yeast, control of pH i is critical for cell survival and is regulated through a concerted moveme...

Lactoferrin Is Broadly Active against Yeasts and Highly Synergistic with Amphotericin B

“…A growing number of studies provide evidence that LF directly interacts with the fungal cell surface and acts on intracellular targets. Various mechanistic actions have been reported, including alteration of cell surface permeability and changes in membrane potential (38); mitochondrial dysfunction, causing intracellular reactive oxygen species accumulation (21,39); and caspase activation and cytochrome c release (18). Similarly, the LF peptides lactoferricin and lactoferrampin are known to act by binding to and disrupting the fungal cell membrane and have been observed to be rapidly internalized by cells (20,40).…”

Identification of Antifungal H + -ATPase Inhibitors with Effect on Plasma Membrane Potential

“…cerevisiae (33) and lactoferrin (>600% increase) in C. albicans (17). Both omeprazole and lactoferrin are described to be Pma1 inhibitors, although omeprazole is not fully capable of inhibiting extracellular acidification (33).…”

The Antifungal Activity of Lactoferrin and Its Derived Peptides: Mechanisms of Action and Synergy with Drugs against Fungal Pathogens

“…In Saccharomyces cerevisiae , it was shown that hLf induces cell death in a mitochondrial and caspase-dependent way that is characterized by caspase activation, ROS accumulation and cytochrome c release (Acosta-Zaldivar et al, 2016). Work by the same group in C. albicans indicated that the proton-translocating ATPase Pma1p, which is a primary contributor to pH regulation in yeasts, was the target of hLf, inducing lethal mitochondrial dysfunction (Andres et al, 2016). …”